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Fig. 8. Nernst plots for Hb Ao in the absence (O) (£1/2 = 85 mV, n 1/2 = 2.0) and presence (A) of 0.2 M KC1 (£1/2 = 125, ni/2 = 1.2). Conditions: 1 m M Ru(NH3)6C13, 0.06 m M heme, in 0.05 M MOPS, pH 7.1, and 20°. Nernst plot for horse Hb (•) (Em = 55mV, «1/2 = 1.7) is also shown. Conditions: 1 m M Ru(NH3)6C13, 0.06 mM heme, in 0.05 M HEPES, pH 7.5, and 20°.

(T-state) quaternary conformation has traditionally explained anion effects on Hb oxygenation.104,105 The presence of heterotropic effectors (e.g., protons, anions, and carbon dioxide) bound at spatially remote sites of the Hb influences the oxygenation process and has been shown to affect the oxidation process in a similar manner.1,3,17,41,69

An example of the influence of the medium composition and globin amino acid sequence on heme redox properties is presented in Fig. 8. A typical Nernst

104 R. E. Benesch, R. Benesch, and C. I. Yu, Biochemistry 8,2567 (1969).

105 R. Benesch and R. E. Benesch, Nature (London) 221, 618 (1969).

plot for Hb Ao (sigmoidal curve) is presented in contrast with a Nernst plot for horse Hb (shifted to more negative potentials with respect to Hb Ao, but keeping its sigmoidal curvature), on the one hand, and for Hb Ao in the presence of 0.2 M KC1 in the medium (flattening of the Nernst plot and £"1/2 shifted to more positive potentials with respect to Hb Ao), on the other hand. This data set illustrates the sensitivity of the Hbs to the presence of anions in solution (allosteric effectors) as well as the importance of the globin structure around the heme site in fine-tuning the redox response of the protein.

We should note that, in the case of the Hbs, the observed sigmoidal Nernst plot is actually influenced by both scenario 1 and scenario 2 presented above, as the a and fi chains of the tetramer are not identical, but cannot be differentiated spectroscopically.2'4'106 If the four chains had exactly the same midpoint potentials, the Nernst plot would show a pure positive cooperativity. How heterogeneous these two a and fi chains are electronically when bound to each other is not known. The extent to which this heterogeneity influences the overall shape of the Nernst plot under various conditions is only speculative. That said, the relative effect of the redox cooperativity typically dominates chain heterogeneity, as illustrated by the observed «1/2 > 1.

Conclusions

The redox behavior of the Hbs and Mbs and the shape of their Nernst plots are sensitive indicators of globin alterations and/or changes in their environment (e.g., medium composition, presence of allosteric effectors, etc). Studying the effect of these variations on the protein oxidation curve has proved to be a useful tool in understanding the basis of the Hb allosteric mechanism on oxidation, but has also provided insight with respect to the mechanism involved in 02 binding.85 The existence of parallels and differences between the oxygenation of the Hbs and their anaerobic oxidation has allowed us to discriminate between the electronic and steric consequences brought about by changes in globin structure.69 We have published results of spectroelectrochemical assays that probed the influence of broad concentration ranges of allosteric effectors (CI-, NC>3~, PO43-, and CIO4") on a number of Hbs that further clarified the impact of these anions on the electronic properties of the active sites of Hbs.1,85 This, in turn, has elucidated a possible mechanism linked to anion-induced restriction of the conformational fluctuations of the Hbs in controlling oxygen affinity.1 These studies have been made possible by the use of a combination between an OTTLE and UV-Vis spectroscopic monitoring, as described in this chapter. The technique and methods of data analysis described here have made a significant impact on the study of redox-active metalloproteins.

106 S. Beychok, I. Tyuma, R. E. Benesch, and R. Benesch, J. Biol. Chem. 242, 2460 (1967).

Acknowledgments

Our work in this area is supported by the NIH (ROl HL58248) and through the support of a Marine/Freshwater Biomedical Center Grant (ESO-19Q8) funded by the NIEHS.

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